Abstract 803: Targeting β-catenin/CBP signaling in OSCC

OBJECTIVES: Oral squamous cell carcinoma (OSCC) is an aggressive malignancy characterized by molecular heterogeneity and locoregional spread associated with high morbidity. Aggressive cancers are thought to arise from populations of cancer initiating cells (CICs) that exhibit the properties of stem cells and drive tumor development, recurrence and resistance to therapy. The transcriptional regulator, β-catenin, has been implicated in OSCC CICs. Nuclear β-catenin has been shown to recruit the chromatin remodeling CREB binding protein (CBP) to drive expression of proliferation and survival genes, as well as genes that maintain stem-like phenotypes. We hypothesized that targeting β-catenin-CBP interaction will inhibit CICs in oral tumors and restore an epithelial phenotype. METHODS: To test tumor aggressive potential of OSCC CICs, we used zebrafish as a model system. We isolated CD44+CD24hiCD29hi cells fom aggressive HSC-3 OSCC cells by FACS and assayed their ability to drive tumor growth and metastases in zebrafish compared to unsorted and CD44+CD24lowCD29low cells. In addition, we examined the role of the β-catenin/CBP axis in the aggressive phenotype of these cells. We also assessed whether the β-catenin/CBP axis affected CICs in tumors from immune competent HPV+ mice. RESULTS: Zebrafish injected with subpopulation of cells co-expressing CD44+CD24hiCD2hi primitive cell surface markers drove rapid tumor growth and metastases, followed by unsorted and sorted CD44+CD24lowCD29low. Treatment of CD44+CD24hiCD29hi cells with a small molecule inhibitor of the β-catenin-CBP interaction, ICG-001, interfered with tumor growth and metastases in zebrafish. Further, ICG-001 inhibited tumor growth in immunocompetent HPV+ murine model. On a cellular level, ICG-001 promoted membrane localization of β-catenin, enhanced E-cadherin adhesion and restored epithelial phenotype. Significantly, ICG-001 gene signatures tracked with reduced overall patient survival in the cancer genome atlas, TCGA. Conclusion: Our studies indicate that the β-catenin/CBP axis promotes OSCC CICs and that ICG-001 may be an effective therapeutic agent for this malignancy.Support: Evans Center for Interdisciplinary Biomedical Research ARC funding AU 5303015 8000000

Predicted values for a) adherens junctions (AJ) and b) adhesivity (<i>σ</i>) when numerically solving RCN model.

<p>The four conditions simulated the experimental conditions: <i>reference condition</i> corresponds to physiological rate of β-catenin/TCF complex formation (<i>i</i>.<i>e</i>. equilibrium constant <i>K</i>_<i>11</i>, <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005007#pcbi.1005007.s005" target="_blank">S1 Table</a>); <i>dysregulated condition</i> corresponds to ICG-001 treatment, modeled as a disruption in β-catenin/TCF complex formation (<i>i</i>.<i>e</i>. rate constant changed to 2×<i>K</i>_<i>11</i>). Wnt “OFF” state is modeled with a total Wnt3a concentration <i>WNT</i>^<i>0</i> <i>= 1 nM</i>, and Wnt “ON” with <i>WNT</i>^<i>0</i> <i>= 28</i>.<i>062 nM</i>.</p

Movement angle is the angle between expected sheet direction (<i>i</i>.<i>e</i>. wound closing or 0°) and PIV determined velocity vectors.

<p>Color bar indicates fraction of all PIV vectors with a particular orientation. <i>Reference condition</i> is DMSO treated and <i>dysregulated condition</i> is ICG-001 treated. CCM stands for control conditioned media; WCM stands for Wnt3a conditioned media.</p

Ranking of processes of RCN in terms of sensitivity of system to corresponding parameter change.

<p>Ranking of processes of RCN in terms of sensitivity of system to corresponding parameter change.</p

Berberis tschonoskiana Regel

原著和名: オホバメギ科名: メギ科 = Berberidaceae採集地: 奈良県 大台ヶ原山 (大和 大台ヶ原山)採集日: 1971/8/27採集者: 萩庭丈壽整理番号: JH034207国立科学博物館整理番号: TNS-VS-98420

MDCK cells were treated with either conditioned media with (WCM) or without (CCM) Wnt3a, and either no inhibitor (DMSO) or ICG-001.

<p>a) Representative immunoblots (IBs) of ABC in total cell lysates (TCL). Images were taken from a single membrane. Quantified intensity values are averages; errors bars represent standard error of the mean (SEM) (N = 4). Full IB with duplicates can be found in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005007#pcbi.1005007.s004" target="_blank">S3 Fig</a> (supplemental). b) IBs for α-catenin and E-cadherin in E-cadherin immunoprecipitates (IP). CCM TLC and WCM TLC represent input, with isotype controls (IP IgG) included. Blots were quantified and normalized to the ICG-001 condition in each activation state. * represents statistically significant difference, p<0.05.</p

E-cadherin mobility shift from treatment of total cell lysates with glycosidases: Endoglycosidase H (EndoH) or Peptide-N-Glycosidase F (PNGaseF).

<p>a) Immunoblots of E-cadherin from cells grown without exogenous Wnt3a (CCM). b) Immunoblots for lysate from cells grown in exogenous Wnt3a (WCM). Cells were grown in the presence of either no inhibitor (DMSO) or ICG-001.</p

Schematic of current notion of the relationship between Wnt/β-catenin signaling, N-glycosylation, and intercellular adhesion.

<p>Image adapted from [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005007#pcbi.1005007.ref025" target="_blank">25</a>].</p

Additional file 1: of Functional and genomic analyses reveal therapeutic potential of targeting β-catenin/CBP activity in head and neck cancer

Document describing Supplemental Materials and Methods, also including Table S1. and Figures S1-S8. (DOCX 2768 kb

Additional file 2: of Functional and genomic analyses reveal therapeutic potential of targeting β-catenin/CBP activity in head and neck cancer

Tabular spreadsheet file containing both table of differential gene expression results comparing ICG-001 treatment (n = 3) versus DMSO (vehicle) control (n = 3) in HSC-3 and CAL27 OSCC cell lines, as well as the ICG-001 treatment gene expression signatures derived thereof (see manuscript “Methods”). (XLSX 4246 kb